Lactoferrin, an iron-binding glycoprotein, is considered an important mediator in host defense against pathogenic organism. The significance of lactoferrin in health and disease has been the subject of several reviews (Sanches L., Calvo M., Brock J H., (1992) Biological role of lactoferrin. Arch Dis Child. 67, 657-661; Lonnerdal B., Iyer S. (1995). Lactoferrin: molecular structure and biological function. Annu. Rev. Nutr. 15, 93-110). Lactoferrin has well-defined, direct antimicrobial activity (Zagulski T, Lipinski P, Zagulska A, Broniek S, Jarzabek Z. Lactoferrin can protect mice against a lethal dose of Escherichia coli in experimental infection in vivo. Br J Exp Pathol. 1989; 70(6): 697-704). It can also be categorized as an immunomediator during inflammatory responses. Lactoferrin is particularly active at mucosal surfaces. Because of its high concentration in human colostrum, lactoferrin has been studied extensively in host defense responses in infants (Brock J. H. Lactoferrin in human milk: its role in iron absorption and protection against enteric infection in the newborn infants. Arch. Dis. Child. 1980; 55, 417-421; Howie P W., Forsyth J S., Ogston S A., Clark A., du V. Florey C. Protective effects of breast feeding. B. M. J. 1990; 300, 11-16). It is theorized that lactoferrin within human milk provides protection against pathogens during newborn adaptation to non-uterine life, and plays a role in rendering breast-fed infants more resistant to the development of microbe-induced gastroenteritis (compared to formula-fed babies). U.S. Pat. No. 4,977,137 of Nichols et al. discloses milk lactoferrin as a dietary ingredient which promotes growth of the gastrointestinal tract of human infants and newborn nonhuman animals immediately on birth. Nichols discusses the use of lactoferrin in the management of short gut syndrome, an anatomical dysfunction.
Lactoferrin has a profound modulatory action on the immune system (Zimecki M., Machnicki M., Lactoferrin inhibits the effector phase of delayed type hypersensitivity to sheep erythrocytes and inflammatory reactions to M. bovis (BCG). Arch Immunol Ther Exp 1994; 42:171-177), it promotes maturation of T cell precursors into immunocompetent helper cells and differentiation of immature B cells to become efficient antigen presenting cells (Zimecki M., Mazurier J., Spik G., Kapp J A. Human lactoferrin induces phenotypic and functional changes in splenic mouse B cells. Immunology 1995; 86:112-127). Lactoferrin is an integral part of the cytokine-induced cascade during insult-induced metabolic imbalance (Kruzel M., Harari Y., Chen Y., Castro A. G. Lactoferrin protects gut mucosal integrity during endotoxemia induced by lipopolysaccharide in mice. Inflammation 2000; 24:33-44). Receptors for Lactoferrin have been identified and characterized on monocytes, B and T cells. Lactoferrin injected intravenously, intraperitoneally, or orally is quickly taken up by cells of the immune system, preferably by cells of the reticuloendothelial-system. Lactoferrin upregulates expression of leukocyte function associated-1 (LFA-1) antigen on human peripheral blood lymphocytes (Zimecki M, Miedzybrodzki R, Mazurier J, Spik G. Regulatory effects of lactoferrin and lipopolysaccharide on LFA-1 expression on human peripheral blood mononuclear cells. Arch Immunol Ther Exp 1999; 47:257-264). As presented in FIG. 1, lactoferrin can modulate the outcomes of acute inflammation, which is fundamentally a protective response to cell injury as disclosed in PCT application number WO 98/50076, entitled “Methods for Preventing and Treating the Insult-Induced Metabolic imbalance in humans and other Animals”, filed May 3, 1997, all of which is incorporated herein by reference.
The role of lactoferrin in modulating both the acute and chronic inflammation is under active investigation. By virtue of high affinity to iron lactoferrin is considered an important component of nonspecific host defense system against various pathogens in humans. However, a high level of lactoferrin in plasma has been suggested to be a predictive indicator of sepsis-related morbidity and mortality (Bayens R D., Bezwoda W R. Lactoferrin and the inflammatory response In: Lactoferrin: Structure and Function, eds. T. W. Hutchens et al., Plenum Press, 1994; pp. 133-141). In addition, progression in chronic inflammatory disorders, such as Alzheimer's disease, or autoimmune disorder such, as multiple sclerosis, seems not to be interrupted by lactoferrin elevation in various physiological fluids. Although, the endogenous production of lactoferrin is increased in these disorders, it is either not sufficient, or does not trigger the pathway(s) of molecular events to aid a defense system against the disorder. It is possible that the exogenous lactoferrin, especially when given orally, transduces different signaling pathways than the endogenous lactoferrin molecule. Consequently, the end effects are different.
Under normal physiological conditions, the rate and magnitude of reactive oxidants formation is balanced by the rate of their elimination. An imbalance between reactive oxidants production and antioxidant defense results in oxidative stress, which may lead to the oxidative cell injury (Touyz R M. “Oxidative stress and vascular damage in hypertension”. Curr Hypertens Rep. 2000; 2(1): 98-105). Oxidative stress can contribute to many diseases including fatigue, sepsis, autoimmune diseases, cancer, neurodegenerative diseases, heart attack and stroke. Transitional metals have been considered as key factors in the oxidative stress. In particular, traces of iron can be detrimental to physiological processes under reactive oxygen conditions. Iron is in a center of the reactive oxygen species control. It has the ability to catalyze two step process known as the Haber-Weiss reaction (FIG. 2). In the first reaction a superoxide molecule reacts with iron (3+) salt to form iron (2+) salt and ground state oxygen. The second reaction is known as the Fenton reaction. In this reaction iron (2+) salt reacts with hydrogen peroxide to form iron (3+) salt, the hydroxyl radical and alcohol.
In normal physiological conditions the production and neutralization of these reactive oxygen species (ROS) depend on the efficiency of key enzymes, including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX). If the process of neutralization of ROS is not efficient, it can contribute to development of oxidative stress (e.g. lipid peroxidation). Although, endogenous lactoferrin participates in these processes at cellular level it is not understood how exogenous lactoferrin would contribute to these molecular events (FIG. 2). Again, based on the recognition that lactoferrin level increases during development of some autoimmune and neurologic conditions, the use of exogenous lactoferrin would not be scientifically justified.
Reactive oxygen species are capable of catalyzing morphological changes to proteins, in both beneficial and non-beneficial ways. The ability of a cell to control these changes in oxidation and resulting protein effects is very important for species survival. Recently, intermediates in the lipid peroxidation process have shown the ability to inactivate and modify proteins. This is an important finding because proteins in biological membranes may become a primary target in radical-induced cell death. Lipid peroxidation is tentatively defined as the oxidative deterioration of polyunsaturated lipids. These fatty acids provide mobility and fluidity to the plasma membrane, properties which are known to be essential for the proper function of biological membranes. The process of lipid peroxidation is a step-wise process with an initiation and subsequent propagation reactions. Iron and other transitional metals help to initiate the process by forming alkeoxy or peroxy radicals upon reaction with oxygen species. The fatty acids are reduced to reactive aldehydes and hydrocarbons. In general, the damaging consequences of lipid peroxidation are expressed as a decrease in the fluidity of the membrane and subsequent increase in its permeability to substances which normally do not pass.
The nervous system, including the brain, spinal cord, and peripheral nerves, is rich in both unsaturated fats and iron (Halliwell. Reactive oxygen species and the central nervous system. J. Neurochem. 1992; 59(5): 1609-23). The high lipid content of nervous tissue, coupled with its high metabolic activity, makes it particularly susceptible to oxidant damage. The high level of brain iron may be essential to oxidative stress via the iron-catalyzed formation of reactive oxygen species.
In the age related disorders that develop over decades, many chemical species as well as pathophysiological conditions are involved. The major threat comes from the oxidative stress. The generation of the reactive oxygen species can lead to immediate damage or death of cells in various tissues (Gutteridge. Hydroxyl radicals, iron, oxidative stress, and neurodegeneration. Ann N Y Acad. Sci. 1994; 738:201-13). There is substantial evidence that oxidative stress is a causative factor in the pathogenesis of major neurodegenerative diseases, including Parkinson's disease (Ebadi M, Srinivasan S K, Baxi M D. Oxidative stress and antioxidant therapy in Parkinson's disease. Prog Neurobiol. 1996; 48(1):1-19), Alzheimer's disease (Markesbery W R, Camey J M. Oxidative alterations in Alzheimer's disease. Brain Pathol. 1999; 9(1):133-46.; Behl Vitamin E and other antioxidants in neuroprotection. Int J Vitam Nutr Res. 1999; 69(3):213-9), and amyotrophic lateral sclerosis (Olanow and Arendash Metals and free radicals in neurodegeneration. Curr Opin Neurol. 1994; 7(6):548-58.; Simonian and Coyle Oxidative stress in neurodegenerative diseases. Annu Rev Pharmacol Toxicol. 1996; 36:83-106) as well as in cases of stroke, trauma, and seizures (Coyle and Puttfarcken. Oxidative stress, glutamate, and neurodegenerative disorders. Science. 1993; 262(5134):689-95.; Facchinetti F, Dawson V L, Dawson T M. Free radicals as mediators of neuronal injury. Cell Mol Neurobiol. 1998; 18(6):667-82) or rheumatoid arthritis, fatigue and cancer (Kovacic P, Jacintho J D. Mechanisms of carcinogenesis: focus on oxidative stress and electron transfer. Curr Med Chem 2001; 8(7):773-96).
Also, there is ample evidence that allergic disorders, such as asthma, rhinitis, and atopic dermatitis, are mediated by oxidative stress (Bowler R P., Capro J D. (2002): Oxidative stress in allergic respiratory diseases J Allergy Clin Immunol. 110:349-56). In fact, the oxidative stress-induced immune hypersensitivity indicates a shift in immunostasis towards the Th2 responses. The Th1/Th2 balance is responsible for coordinating the immune system and become very important during aging processes, including the development of autoimmune, neurodegenerative and immune hypersensitivity disorders.
Although, considerable data from in vitro experiments indicate several physiological roles for lactoferrin, there is no firm evidence concerning its actual physiological function from in vivo studies. For example, in a review by Roy D. Byens and Werner R. Bezwoda entitled “Lactoferrin and the inflammatory response” and published in the book: Lactoferrin: Structure and Function, pp 133-141, (1994), a relationship between plasma lactoferrin and granulocyte activity in sepsis is mentioned. However, the biological function of the significant amounts of lactoferrin in plasma of septic patients is as yet not completely understood.
Similarly, marked elevation of lactoferrin has been noted in the cerebrospinal fluid of patients with acute cerebrovascular lesions and other pathological lesions in variety of neurodegenerative disorders (Penco S, Villaggio B, Mancardi G, Abbruzzese M, Garre C. A study of lactoferrin and antibodies against lactoferrin in neurological diseases. Adv Exp Med Biol. 1998; 443:301-40). Based on this observation the use of exogenous lactoferrin in patients who overexpress its own lactoferrin would not be scientifically justified.
In another review entitled “The role of lactoferrin as an anti-inflammatory molecule” by Bradley E. Britigan, Jonathan S. Serody, and Myron S. Cohen and published in the book: Lactoferrin: Structure and Function, pp 143-156, (1994), the role of lactoferrin in inflammation is suggested to be played at two different levels: (i) as an antioxidant, capable of binding free iron, and (ii) as an endotoxin scavenger, capable of reducing lipopolysaccharide (LPS)-induced toxicity.
In yet another article entitled “Lactoferrin in infant formulas: effect on oxidation”, by Satue-Gracia M T, Frankel E N, Rangavajhyala N, German J B., and published in J Agric Food Chem. 2000; 48(10):4984-90, authors emphasize the ability of lactoferrin to control oxidation in infant formulas.
Relevant patents are also silent as to the use of lactoferrin for prevention or therapy of autoimmune or neurodegenerative disorders in humans and animals. U.S. Pat. No. 5,240,909 of Nitsche relates to the use of lactoferrin as an agent for the prophylactic and therapeutic treatment of the toxic effects of endotoxins. Nitche discloses that the lactoferrin used according to his invention has the ability to neutralize endotoxin and must have bound to it either iron or another metal to be effective. U.S. Pat. No. 5,066,491 of Stott et al. encompasses a method of disease treatment utilizing a therapeutically effective product produced from ordinary milk whey.
Despite large number of studies on lactoferrin, there is no disclosure that it can function as a mediator to reduce the debilitating conditions in the autoimmune, neurodegenerative and immune hypersensitivity disorders such as Alzheimers, Parkinson's, multiple sclerosis, rheumatoid arthritis, cancer, allergy, stroke or fatigue. The knowledge about endogenous lactoferrin is not supporting the clinical effects of exogenous lactoferrin as found in the present invention. For example, autoantibodies to lactoferrin are commonly found in many autoimmune disorders, including multiple sclerosis (Penco S, Villaggio B, Mancardi G, Abbruzzese M, Garre C. A study of lactoferrin and antibodies against lactoferrin in neurological diseases. Adv Exp Med Biol. 1998; 443:301-40) and rheumatoid arthritis (Locht H, Skogh T, Kihlstrom E. Anti-lactoferrin antibodies and other types of anti-neutrophil cytoplasmic antibodies (ANCA) in reactive arthritis and ankylosing spondylitis. Clin Exp Immunol. 1999; 117(3):568-73). In fact, the presence of these antibodies has been suggested to be used as marker for the inflammatory disorders. Based on this observation the use of lactoferrin in patients with autoantibodies to lactoferrin would not be scientifically justified. According to present invention, exogenous lactoferrin has been found to reduce the symptoms of autoimmune, neurodegenerative and immune hypersensitivity disorders.